Construction and expression of a flavocytochrome b5 chimera. J Biol Chem 1994 May 06;269(18):13375-81
Date
05/06/1994Pubmed ID
8175767Scopus ID
2-s2.0-0028229514 (requires institutional sign-in at Scopus site) 9 CitationsAbstract
A gene has been constructed coding for a chimeric flavocytochrome b5 protein that comprises the soluble domain of rat hepatic cytochrome b5 as the NH2-terminal portion of the chimera and the flavin-containing domain of spinach assimilatory NADH:nitrate reductase as the C terminus. The chimeric protein has been expressed in Escherichia coli and purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose, anion-exchange chromatography, and fast protein liquid chromatography gel filtration with an estimated molecular mass of 43 kDa from polyacrylamide gel electrophoresis. Visible and fluorescence spectroscopy indicated the purified protein contained both a b-type cytochrome and FAD prosthetic groups. The chimeric hemoflavoprotein immunologically cross-reacted with both anti-rat cytochrome b5 and anti-spinach nitrate reductase polyclonal antibodies, indicating the conservation of antigenic determinants from both native domains. NH2-terminal and internal amino acid sequencing of the native and CNBr-digested protein confirmed the presence of peptides derived from both the heme- and flavin-binding portions of the sequence which were identical to the deduced amino acid sequence. The chimera exhibited both NADH: ferricyanide reductase and NADH:cytochrome c reductase activities with Vmax values of 88 and 37 mumol of NADH consumed per min/nmol of heme (mu = 0.05 and pH 7.0) and Km values of 2.1, 32, and 1.4 microM for NADH, ferricyanide, and cytochrome c, respectively. This work represents the first successful bacterial expression of a mammalian-plant chimeric metalloflavoprotein. The chimera exhibited properties extremely similar to those of the native cytochrome b5 heme and spinach nitrate reductase FAD components.
Author List
Quinn GB, Trimboli AJ, Barber MJAuthor
Anthony J. Trimboli PhD Assistant Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceAnimals
Base Sequence
Chromatography, Gel
Cloning, Molecular
Cytochromes b5
DNA, Recombinant
Electrophoresis, Polyacrylamide Gel
Escherichia coli
Genetic Vectors
Immunochemistry
Kinetics
Liver
Molecular Sequence Data
Nitrate Reductase (NADH)
Nitrate Reductases
Rats
Recombinant Fusion Proteins
Sequence Alignment
Spectrum Analysis
Vegetables